molecules Review Conservation of the Keap1-Nrf2 System: An Evolutionary Journey through Stressful Space and Time Yuji Fuse 1,2 and Makoto Kobayashi 1,* 1 Department of Molecular and Developmental Biology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan; [email protected] 2 Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-8575, Japan * Correspondence: [email protected]; Tel.: +81-29-853-8457 Academic Editors: Luciano Saso, László Dux, Grzegorz Wegrzyn and Tamás Csont Received: 9 February 2017; Accepted: 6 March 2017; Published: 9 March 2017 Abstract: The Keap1-Nrf2 system is an evolutionarily conserved defense mechanism against oxidative and xenobiotic stress. Its regulatory mechanisms, e.g., stress-sensing mechanism, proteasome-based regulation of Nrf2 activity and selection of target genes, have been elucidated mainly in mammals. In addition, emerging model animals, such as zebrafish, fruit fly and Caenorhabditis elegans, have been shown to have similar anti-stress systems to mammals, suggesting that analogous defense systems are widely conserved throughout the animal kingdom. Experimental evidence in lower animals provides important information beyond mere laboratory-confined utility, such as regarding how these systems transformed during evolution, which may help characterize the mammalian system in greater detail. Recent advances in genome projects of both model and non-model animals have provided a great deal of useful information toward this end. We herein review the research on Keap1-Nrf2 and its analogous systems in both mammals and lower model animals. In addition, by comparing the amino acid sequences of Nrf2 and Keap1 proteins from various species, we can deduce the evolutionary history of the anti-stress system. This combinatorial approach using both experimental and genetic data will suggest perspectives of approach for researchers studying the stress response. Keywords: anti-stress system; Drosophila Cnc; evolutionary history; Hydra Nrf; Keap1; mouse; Nrf2; C. elegans Skn-1; yeast Yap1; zebrafish 1. Introduction From birth, animals are destined to fight against a variety of stressors that disrupt their homeostasis. All animal species must cope with oxidative stress generated by their own metabolism. They were also forced to evolve detoxifying systems in case of accidental encounters with toxic chemicals in the environment. Animals could not have prospered without the anti-stress mechanisms evolved by their ancestral species. Mammals inherited from their ancestors the Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system, which is a defense system that confers protection against a wide spectrum of stressors, including oxidative and chemical stress. Recent advances have revealed that the system is related to a number of human diseases, such as cancer, neurodegenerative diseases and diabetes mellitus [1–3], and many researchers are investigating potential medical applications. Although the Keap1-Nrf2 system has mainly been studied using mice and human cells, increasing evidence suggests that orthologous systems exist in lower vertebrates, such as zebrafish [4], and even in Drosophila [5], which will be promising tools for accelerating the study of Nrf2. Molecules 2017, 22, 436; doi:10.3390/molecules22030436 www.mdpi.com/journal/molecules Molecules 2017, 22, 436 2 of 22 The advantages of lower-model animals are not limited to their usefulness in the laboratory and few ethical problems. Recent improvements in genome projects have provided high-quality genomic sequences, facilitating the development of lower-model organisms and enabling us to assess specific biological functions from an evolutionary point of view. In addition to classic model animals, genomic information from non-model animal species has been increasingly accumulated. This information prompted us to track the evolutionary path of proteins of interest. In the present review, we will highlight the evolution of the Keap1-Nrf2 system and attempt to identify the unique characteristics of the mammalian Nrf2 system by reviewing the evolutionary history of the conserved anti-stress mechanism. 2. Overview of the Keap1-Nrf2 System 2.1. Transcription Factor Nrf2 and Its Function Nrf2 was discovered as a homolog of nuclear factor-erythroid 2 p45 (NF-E2), which plays an essential role in the transcriptional regulation of the β-globin gene [6,7]. Unlike NF-E2, the function of this newly-discovered transcription factor was not related to hematopoiesis; Nrf2-knockout mice did not show any obvious phenotype and were normally grown and fertile with no anemia [8–10], suggesting that Nrf2 regulates a different battery of genes from NF-E2. The function of Nrf2 was first reported by Itoh et al. [11], who noticed the similarity between the NF-E2 binding sequence and antioxidant responsive element (ARE). This regulatory sequence is usually found upstream of genes encoding phase II detoxifying enzymes and had been known to regulate the induction of these genes [12,13]. The down-regulation of the expression of phase II enzymes in Nrf2-knockout mice indicated that Nrf2 regulated the global transcription of phase II enzymes through ARE-dependent signals [11]. The main function of phase II enzymes is to detoxify the highly reactive intermediate metabolites generated by phase I reactions and accelerate the excretion of toxic xenobiotics [14]. Benzo[a]pyrene is a well-studied pro-carcinogen that forms a highly reactive intermediate after phase I metabolism and is detoxified by phase II reactions. The loss of Nrf2 was suspected to potentially weaken phase II metabolism and enhance the carcinogenicity of benzo[a]pyrene. This was experimentally demonstrated by showing the high susceptibility of Nrf2-deficient mice to benzo[a]pyrene-induced tumor formation, suggesting that Nrf2 is indispensable for intact phase II metabolism [15–17]. Later studies revealed that the Nrf2 system also regulates phase III xenobiotic transporters [18,19] as well as phase I-related genes [20,21], suggesting that Nrf2 is involved in the entire process of xenobiotic metabolism. In addition, as ARE sequences were also found upstream of antioxidative genes, such as heme oxygenase 1, Nrf2 was shown to be the master regulator of the oxidative stress response [22]. The mode of action of many toxic chemical stressors is the generation of reactive oxygen species; therefore, Nrf2 plays an important role in the defense against various chemical-derived stresses, such as diesel exhaust, peroxide, heavy metals and other electrophilic compounds [23–25]. The Nrf2-dependent induction of xenobiotic metabolism and antioxidant system likely contributed greatly to animal evolution in a rapidly changing environment. The Nrf2 system is also activated by endogenous cues, such as endoplasmic reticulum (ER) stress, and confers defense against such stress [26,27]. This protection may be partly due to the Nrf2-dependent induction of proteasome subunits [28,29], which destroy unfolded proteins accumulated in the cell. Another endogenous Nrf2-activating signal is the disruption of autophagy [30], which implies that the Nrf2 system can be activated by disorders of protein turnover. Numerous studies have shown that these internal stresses are related to various diseases; as such, Nrf2 has attracted attention as a potential therapeutic target. By contrast to these beneficial effects on human health, adverse effects of the Nrf2 system have also been reported: Nrf2 is constitutively activated in cancer cells and confers resistance against chemotherapy [31–34]. Surprisingly, Nrf2 activates the pentose phosphate pathway and remodels Molecules 2017, 22, 436 3 of 22 cellular metabolism, which enhances cancer cell proliferation [35,36]. These findings may support the advent of Nrf2-blocking therapy for cancer patients. 2.2. Regulatory Mechanism of Nrf2-Dependent Gene Induction Molecules 2017, 22, 436 3 of 21 Research on the Nrf2 activation mechanism has greatly progressed since a partner protein of 2.2. Regulatory Mechanism of Nrf2-Dependent Gene Induction Nrf2, Keap1, was discovered (Figure1A). Keap1 was discovered as a protein that binds directly to Nrf2 andResearch negatively on the regulates Nrf2 activation the transcriptionalmechanism has greatly activity progressed of Nrf2 since [37 a]. partner As Keap1 protein isof theNrf2, adaptor Keap1, was discovered (Figure 1A). Keap1 was discovered as a protein that binds directly to Nrf2 and protein for the ubiquitin ligase, Nrf2 is ubiquitinated in a Keap1-dependent manner and degraded negatively regulates the transcriptional activity of Nrf2 [37]. As Keap1 is the adaptor protein for the by proteasomeubiquitin systemligase, Nrf2 [38– is40 ubiquitinated], which keeps in a theKeap1-dependent Nrf2 protein manner level lowand underdegraded unstressed by proteasome conditions. Nrf2-activatingsystem [38–40], compounds which keeps inhibit the the Nrf2 function protein level of Keap1 low under by attacking unstressed highly conditions. reactive Nrf2-activating cysteine residues and stabilizecompounds Keap1-Nrf2 inhibit the binding. function Deof Keap1 novo-synthesized by attacking highly and reactive accumulated cysteine residues Nrf2 translocates and stabilize into the nucleusKeap1-Nrf2 and binds binding. to ARE De with novo-synthesized its heterodimeric and accu partner,mulated small Nrf2 translocates musculoaponeurotic into the nucleus fibrosarcoma and (small Mafs:binds